Fungal infections have become a significant and increasing cause of severe illness and death. Candida glabrata is emerging as a lethal fungal pathogen, contributing significantly to the mortality already caused by the prevalent pathogen, Candida albicans. Unfortunately, C. glabrata is inherently resistant to several of the available antifungal therapeutics, including amphotericin B and the azole compounds. Therapeutics to treat C. glabrata infections are critically necessary. Furthermore, since systemic fungal infections often proceed rapidly toward death and formal diagnosis requires critical time, an ideal therapeutic would also be a broad spectrum antifungal agent that is also effective against the primary fungal pathogen, C. albicans. Building on previous success to target dihydrofolate reductase (DHFR) in pathogenic organisms, we have synthesized a class of versatile DHFR inhibitors. Several of an initial series of our inhibitors exhibit strong potency against the fungal DHFR enzymes, good selectivity against the mammalian enzyme, good antifungal activity in cultures of the organisms and little appreciable mammalian cell toxicity. We have also crystallized one of our inhibitors bound to C. glabrata DHFR and determined the structure to 1.6 E resolution. In the first aim of this application, we propose to use a structure-guided approach to design and synthesize new analogs that potently and selectively target C. glabrata DHFR. In a second parallel aim, we will determine efficacy of the compounds in cells and animals, elucidate resistance mechanisms and determine structures of resistant enzymes. In a third aim, we will evaluate these inhibitors against C. albicans and determine crystal structures of potent and selective inhibitors with C. albicans DHFR. In a fourth aim, we propose to generate a potent and selective broad spectrum inhibitor that exhibits excellent antifungal activity against both Candida species and maintains a lack of toxicity against mammalian cells. PUBLIC HEALTH RELEVANCE: Systemic fungal infections have become a significant and devastating cause of severe illness and mortality. Infections caused by Candida glabrata have become increasingly prevalent and are resistant to many of the standard antifungal therapeutics. In this proposal we aim to use a structure-guided approach to synthesize inhibitors of dihydrofolate reductase (DHFR) effective against C. glabrata and to evaluate those inhibitors in cells and animals. We also aim to create a potent and selective broad spectrum inhibitor of C. glabrata and C. albicans DHFR that can be used prior to formal diagnosis of the Candida spp. infection.